This invention was made with government support under Contract No. GM37277 awarded by the National Institute of General Medical Sciences of the United States Department of Health and Human Services. The government has certain rights in this invention.
The invention is in the field of electrophoresis. It is of particular interest in terms of its applications in genetic engineering and molecular biology.
The invention, which is a new kind of pulsed field electrophoresis, makes it possible to determine the effective field angle of the particles undergoing separation by pulsing routine, rather than by placement of an electrode array. In addition, the present electrophoretic technique makes it possible for the first time to translate particles undergoing separation incrementally through a gel matrix. Additionally, the invention makes it possible to separate with a high degree of resolution and at high speeds larger particles (molecules) than those capable of resolution using conventional pulsed field electrophoresis, and to separate particles in a selectable narrow range of sizes (molecular weight).
Electrophoresis in which particles such as a mixture of macromolecules are moved, for example, through a gel matrix, by an electric field, is a widely-used technique for qualitative analysis and for separation, recovery and purification. It is particularly important in the study of chromosomes, proteins and nucleic acids.
It is known that the DNA molecules which make up chromosomes exist in solution as random coils, which resemble loose bunches of yarn moving about in the thermal environment of the solution. Such a DNA coil 10 is shown in FIG. 1. The hydrodynamic radius of a large DNA molecule, as measured by sedimentation or light scattering, can be enormous. For example, a 600 kb (kb=kilobase pair, a unit of length for nucleic acids consisting of 1,000 nucleotide pairs) DNA molecule is approximately 4.times.10.sup.-4 cm in diameter, while an agarose gel pore has an approximate diameter of 1.times.10.sup.-5 cm.
During electrophoresis, the large random coils of the DNA molecules must be deformed by an electric field in order to pass through a gel matrix 12 in the form of a compressed aligned coil 14. Using the technique of the disclosed invention as described below, it has now been shown for the first time that during electrophoresis, a DNA coil 10 compresses into a series of blobs 16 as shown in FIG. 2. The blobs 16 each contain as much as 10 kb of DNA. A train of blobs 16 migrates in an aligned fashion through the gel matrix 12. The degree of alignment of the blob train is determined by the electric field strength and varies as E.sup.2.
Previous to now, there has not been disclosed an effective method for the electrophoretic separation of very large particles. For example, using previously known electrophoretic techniques, the size of the largest DNA molecule routinely handled is 2,000 kb, although a maximum size of 10,000 kb is possible.
Using conventional gel electrophoresis, DNA molecules up to approximately 50 kb, or the size of lambda bacteriophage DNA, may be separated. Because of its relatively large pore size (1.times.10.sup.-5 cm, as noted above) and because it does not bind DNA molecules, agarose is the matrix of choice. Separation is usually performed in a horizontal or vertical gel and the migration of molecules is size dependent up to approximately 50 kb. Above this size, DNA molecules tend to run with a common mobility; that is, all large DNA molecules run together in a gel.
Although particles of higher mass (i.e., up to approximately 600 kb) can be resolved by reducing the gel concentration to as low as 0.035% and reducing field strength, there are drawbacks to this method. Most notably, the dramatic reduction in gel concentration adversely affects resolution, and makes experimental conditions difficult to control. In addition, an electrophoretic run using a reduced gel concentration and field strength can take a week or more to complete.
Pulsed field electrophoresis, developed by the present inventor and described in U.S. Pat. No. 4,473,452 (the disclosure of which is hereby incorporated by reference and relied upon), improves the separation of large DNA molecules in a gel matrix. According to this technique, deliberately alternated electric fields, rather than the uniform fields sought in previously known electrophoretic methods, are used to separate particles. More particularly, particles are separated using electric fields of equal strength which are transverse to each other, which alternate between high and low intensities out of phase with each other at a frequency related to the mass of the particles and which move the particles in an overall direction transverse to the respective directions of the fields. (It should be noted here that the term "transverse" as used herein is not limited to an angle of, or close to, 90.degree., but includes other substantial angles of intersection.)
Thus, an electric field is pulsed between alternate sets of electrodes, forcing the periodic relaxation and reorientation of the particles undergoing separation. Pulse times are approximately equal to the molecular reorientation times for achieving molecular size resolution. When the direction of the electric field is changed, small particles quickly orient themselves and start a new migration along the new path. Larger particles, on the other hand, remain substantially immobile until they are reoriented in the direction of the electric field. Then, they too begin to move in the new direction. By that time, the smaller particles will have moved ahead of them. Thus, separation by size occurs.
Using the technique of the present invention, it is now possible for the first time to directly determine the dynamic molecular conformation of individual particles, for example, DNA molecules, in a gel matrix as oriented by a specially modulated electric field. Using fluorescence microscopy/image processing as described below in Example 5, it has been determined that during pulsed field electrophoresis, the blob train of a DNA molecule orients with the applied electric field in a very complicated manner and during this process, electrophoretic mobility is retarded until alignment is complete. Upon field direction change, the blob train moves in several new directions simultaneously (i.e., the blobs appear to be moving somewhat independently). Eventually, some part of the blob train dominates in reorienting with the applied field and pulls the rest of the blobs along its created path through the gel matrix. The time necessary for complete blob train alignment varies directly with size; i.e., a 10 mb (1 mb=1,000 kb) molecule requires one hour to reorient, while a 10 kb molecule requires only ten seconds, using similar field strengths.
The length of the pulse in each direction determines the size of the molecules that will separate from each other. A fast pulse will cause small molecules to separate, while a slow pulse will cause large molecules to separate. Thus, changing the pulse time selectively modulates the electrophoretic mobility of the molecules in a size dependent manner.
For a given molecular weight, electrophoretic mobility is modulated by varying the pulse time. The minimum measured mobility is obtained when the pulse time is equal to the orientation time. This is called the resonance time. Because the blob train orientation process retards mobility during orientation, the maximum amount of retardation occurs when the pulse time equals the orientation time. However, if the pulse time exceeds the orientation time, then only a portion of the mobility suffers the orientation retardation effects, resulting in increased mobility. Finally, when the pulse time is less than the orientation time, the blob train does not have sufficient time to fully orient, which results in an intermediate orientation as determined by the two applied fields. Thus, the amount of orientation that a blob train requires to fully orient with a given applied field is minimized, and since orientational effects retard velocity, the resulting mobility is increased relative to the resonance time induced mobility, but less than conventional gel electrophoresis.
The angle between applied fields in pulsed field electrophoresis is known to have a profound effect upon separation, although the molecular basis for separation dependence on field angle (the angle measured between two alternately applied fields) remains obscure. Molecules below about 600 kb resolve fairly well when the field angle is 90.degree.. However, obtuse angles give the best separations for molecules larger than about 650 kb.
Schwartz and Cantor (Cell, 37:67-75, 1984) designed pulsed electrophoresis apparatus utilizing field gradients. A field gradient is a variation of field strength over a measured distance, i.e., volts/cm.sup.2. As a consequence of using field gradients, the angle between the two applied fields varied from 90.degree. to 135.degree.. The average angle a molecule experiences in this apparatus is obtuse. Although resolution was adequate, the field gradients caused extensive distortion of the DNA banding patterns, wherein the DNA did not migrate in straight lanes, but in curvilinear paths, which made it difficult to compare adjacent bands.
Researchers later eliminated band distortion by using two homogeneous (ordinary) electrical fields oriented at 120.degree. to each other. As with the above mentioned field gradient approach, both instruments are capable of resolving up to approximately 10,000 kb.
Lane distortions can also be eliminated using Field Inversion Gel Electrophoresis (FIGE) or Reverse Field Electrophoresis (RFE), wherein the field is periodically reversed through a 180.degree. angle. This method differs from other variations of pulsed field electrophoresis in that the alternate pulse times are not equal; i.e., the forward pulse times are longer than the backward pulse times. The range of molecular weights that can be resolved using this method is limited to approximately 1,200 kb.
Thus, despite the fact that pulsed field electrophoresis has provided improved results in the electrophoretic separation of particles, it has been known for some time that the method suffers from important limitations, e.g., human chromosomally sized DNA molecules (100 mgb; 1 mgb=1,000 kb) cannot be resolved using pulsed field electrophoresis. Although the need to overcome the limitations of the prior art has also been known for some time, no previous proposals have been put forward which successfully overcome such limitations.